Figure 5

One hour treatment with AZD8055 affects pER _ser167 protein expression in TamR and MCF7-X cells. Western blot of 70% confluent TamR and MCF7-X cells treated for one hour with AZD8055 (25 to 100 nM). Images show blots probed for pER (s118 and s167), total ER, mTOR phosphorylation on s2481 and s2448 to demonstrate mTOR inhibition and actin to indicate equal loading. The accompanying bar charts show densitometric analysis of western blot data normalised to actin. pER_ser167 but not pER_ser118 protein was inhibited in MCF7-X and TamR cells. Blots are representative of two independent experiments (A). Accompanying immunocytochemical images confirm the effect of AZD8055 on pER_ser167 but not pER_ser118 protein expression in TamR and MCF7-X cells. Immunocytochemistry was performed on TamR and MCF7-X cells fixed after treatment +/- AZD8055 (100 nM) for one hour and cells were stained with antibodies for pER_ser118 and pER_ser167(B). At least five fields of view were examined for staining and representative images (x20) shown. Results are shown from at least three independent experiments.